Immunofluorescence Assay (IFA)

Immunofluorescence assays (IFA) were conducted as previously described (Ye et al., 2015). Cells were seeded in 24-well plates at a confluence of 60–70%. After adherence, the cells were infected with EV71 at an MOI of 1 for 1 h, with rocking every 15 min. After absorption, the inoculum was removed, and medium with remdesivir at varying concentrations was added. At 24 h post infection (hpi), the cells were subjected to an IFA following an established protocol. Briefly, cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% Triton X-100. Cells were incubated with primary antibody (J2) at 4°C overnight and with fluorescein-conjugated secondary antibody at 37°C for 2 h. Hoechst 33258 was used to stain cell nuclei, and the samples were imaged with an BX60 fluorescence microscope (Olympus, Tokyo, Japan).